The study city include c. 2 kilometer dos out of kinds-steeped inactive calcareous grassland inside west Estonia (°Letter, °E). This new grasslands setup typically because of long-identity thorough government, significantly grazing by residential animals, get together hay and you may reducing shrubbery and woods to own strength. , 1999 ). The brand new grasslands traditionally had a max juniper (Juniperus communis) protection out of 29%, but they are today even more overgrown that have Scots pine (Pinus sylvestris; around 80% cover).
Testing is actually used from inside the within internet sites where continuously managed (mown) unlock grassland really stands (GR) was in fact in the middle of really stands off differing degrees of over growing: transformation grasslands (TR) and you will young pine woods (FO). The 3 successional amounts (GR, TR, FO) mode a temporal sequence from regeneration series covering c. 60 yr, and therefore are characterised by the a gradual overgrowth out-of discover grasslands of the shrubs and you may trees, that’s normal for given up calcareous grasslands on studies area (Neuenkamp mais aussi al., 2016 ; Support Recommendations Fig. S1). From inside the transitional grasslands administration ceased c. 30–thirty-five yr ahead of sampling and grasslands were partly overgrown from the pine and you may juniper; younger pine forest was previous grasslands in which administration stopped c. sixty yr in advance of testing and you will that happen to be completely overgrown from the pine and you may juniper. A web site is actually recognized as a location in which all the around three successional values had been receive contained in this fifty m of any most other (GR–TR–FO). Entirely, twenty-five sites was sampled. An arbitrarily receive step one ? step 1 m area is established in each successional stage (GR, TR, FO) of any web site, resulting in a maximum of 75 plots (25 sites ? around three successional values). Another attributes was indeed revealed per plot: the field covering bush people, Have always been yeast teams (in plant roots, and you may rhizosphere ground) and you may environmental parameters.
For each and every 1 ? step 1 yards plot, most of the vascular plant kinds regarding soil-level people was in fact registered as well as their variety is estimated while the commission coverage (Peet & Roberts, 2013 ). Likewise, commission cover quotes out-of shrubbery and you may woods contained in this a good ten ? 10 m town as much as for each and every area was indeed registered. Hereafter, a floor-coating plant community is called ‘bush community’ and all of analyses is actually conducted with this particular neighborhood unless expressed if you chatango don’t.
A good ten ? 10 ? ten cm surface key is actually gathered throughout the heart of each spot and you will bush root have been split from the floor, dehydrated to have twenty four h at the fifty°C and you will held inactive for further unit data from options-colonising Am fungus. Have always been yeast range in plant sources was then analysed from blended means samples. Origins within the each test was indeed crushed having fun with h2o nitrogen and you may thoroughly combined as described from the Garcia de Leon ainsi que al. ( 2016 ). A great subsample out of 70 milligrams each and every blended means test is actually useful for molecular analyses.
Plant teams was revealed during the a more impressive spatial size compared to Are fungal communities, as this scale would be to around mirror the brand new plant resources society seized because of the a great ten ? ten ? ten cm floor key. Small-level plant assortment for the grasslands was higher below ground than just more than floor (Hiiesalu ainsi que al., 2014 ), partially because the roomy sources program and you can much time rhizomes many grassland vegetation hook up the latest origins within a localized soil key with over-crushed areas increasing several ).
To characterise AM fungal diversity in the soil, 10 g of soil was collected from each soil core, dried with silica gel and stored airtight at room temperature. A second subsample of 10 g of soil was collected from each soil core for measuring the concentration of an AM fungal neutral lipid fatty acid fungal abundance in the soil. This second soil sample was freeze-dried for 15 h at ?80°C and stored airtight at room temperature. The remainder of each soil core was air-dried for analysis of soil chemical properties (pHKCl, Ntot, P, K, Mg, Ca, Corg) following International Organization for Standardization (ISO) protocols (see Methods S1).